Abstract PO024: Combination therapy with PXS-5505 improves chemotherapeutic efficacy and reduces myeloid immune suppression in murine cholangiocarcinoma – a novel therapeutic strategy for clinical translation
Authors: Paul R Burchard, Luis I Ruffolo, Nicholas A Ullman, Yatee Dave, Benjamin Dale, Mary Georger, Rachel Jewell, Nabeel Badri, Brian A Belt, David C Linehan, Roberto Hernandez-Alejandro
Journal: Clinical Cancer Research (2022)

Abstract Introduction: Despite growing prevalence, 5-year survival for cholangiocarcinoma remains poor due to an immunosuppressive fibroinflammatory stroma, featuring abundant collagen and cancer-associated fibroblasts. Lysyl oxidases (LOX) consist of 5 secreted enzymes that catalyze collagen cross-linking, resulting in increased tumor stroma density and facilitate immune evasion. Here, we present our findings of pan-LOX inhibition with a novel orally dosed agent (PXS-5505) in a murine model of CCA. Methods: Immunohistochemistry (IHC) analysis was performed on a human tissue macro-array of CCA. Mice that develop spontaneous CCA (KPPC) or orthotopically injected with a syngeneic cell line derived from a KPPC tumor were screened for disease onset and progression with ultrasonography (U/S). Groups of KPPC mice or mice bearing orthotopic tumors were enrolled into treatment cohorts of chemotherapy with or without PXS-5505 to evaluate survival and to perform fixed time point studies with treated tumors including assessment of intratumoral interstitial fluid pressure (IFP), IHC analysis of tumor markers, chemotherapy drug perfusion into tumors by mass spectrometry, and flow cytometry analysis of tumor single cell suspensions for stromal and immune cell populations. Results: IHC analysis demonstrated significantly elevated LOX expression in both human and KPPC CCA (p&amp;lt;0.0001 and p=0.0025, respectively) compared to normal liver controls. High LOX expression was associated with worse disease-free (p=0.02) and overall (p=0.03) survival in resected human CCA specimens. KPPC mice treated with the combination of chemotherapy plus PXS-5505 demonstrated delayed tumor growth (p&amp;lt;0.0001) and ascites onset (p=0.005) with improved survival (median 77 vs. 59 days, p=0.001) compared to chemotherapy alone. In addition, combination therapy reduced IFP (p=0.002) and was associated with increased mean blood vessel area (p&amp;lt;0.0001) and increased concentration of intratumoral chemotherapy levels (p=0.02) compared to chemotherapy alone. Subsequent IHC revealed increased cell death through cleaved caspase-3 (p=0.0003) and damage-associated molecular pattern (DAMP) release through HMGB1 (p=0.0004). Flow cytometry analysis of tumors treated with combination therapy demonstrated reduced prevalence of tumor-infiltrating monocytic and granulocytic myeloid-derived suppressor cell populations (p=0.002 and p=0.01, respectively) and tumor associated macrophages (TAMs; p=0.02). There were increased CD4+ (p=0.01) T-cells with a reduced proportion of Treg+CD4+ (p&amp;lt;0.0001) T-cells. The ratio of CD8:Treg was significantly elevated in the combination therapy cohort (p=0.001) and depletion of T-cells in orthotopic tumors abrogated the improved survival with combination therapy. Conclusions: LOX is elevated in human CCA and its expression correlates with prognosis following resection. Combination therapy with PXS-5505 delays tumor progression and improves survival in murine CCA models. Thus, chemotherapy combined with PXS-5505 represents a novel therapeutic strategy for treating CCA. Citation Format: Paul R Burchard, Luis I Ruffolo, Nicholas A Ullman, Yatee Dave, Benjamin Dale, Mary Georger, Rachel Jewell, Nabeel Badri, Brian A Belt, David C Linehan, Roberto Hernandez-Alejandro. Combination therapy with PXS-5505 improves chemotherapeutic efficacy and reduces myeloid immune suppression in murine cholangiocarcinoma – a novel therapeutic strategy for clinical translation [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO024.